Cholesterol Reduces Pardaxin's Dynamics—A Barrel-Stave Mechanism of Membrane Disruption Investigated by Solid-State NMR

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Biochimica et Biophysica Acta 1798 (2010) 223–227

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Cholesterol reduces pardaxin's dynamics—a barrel-stave mechanism of membrane disruption investigated by solid-state NMR

Ayyalusamy Ramamoorthy ⁎, Dong-Kuk Lee 1, Tennaru Narasimhaswamy 2, Ravi P.R. Nanga

Biophysics and Department of Chemistry, University of Michigan, Ann Arbor, MI 48109-1055, USA

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Article history: While high-resolution 3D structures reveal the locations of all atoms in a molecule, it is the dynamics that Received 9 June 2009 correlates the structure with the function of a biological molecule. The complete characterization of Received in revised form 11 August 2009 dynamics of a membrane protein is in general complex. In this study, we report the inﬂuence of dynamics on Accepted 15 August 2009 the channel-forming function of pardaxin using chemical shifts and dipolar couplings measured from 2D Available online 28 August 2009 broadband-PISEMA experiments on mechanically aligned phospholipids bilayers. Pardaxin is a 33-residue antimicrobial peptide originally isolated from the Red Sea Moses sole, Pardachirus marmoratus, which Keywords: Dynamic functions via either a carpet-type or barrel-stave mechanism depending on the membrane composition. Our Pardaxin results reveal that the presence of cholesterol signiﬁcantly reduces the backbone motion and the tilt angle of Membrane orientation the C-terminal amphipathic helix of pardaxin. In addition, a correlation between the dynamics-induced Barrel-stave heterogeneity in the tilt of the C-terminal helix and the membrane disrupting activity of pardaxin by the Antimicrobial peptide barrel-stave mechanism is established. This correlation is in excellent agreement with the absence of hemolytic activity for the derivatives of pardaxin. These results explain the role of cholesterol in the selectivity of the broad-spectrum of antimicrobial activities of pardaxin. © 2009 Elsevier B.V. All rights reserved.

1. Introduction significant need for biophysical studies to identify the roles of membrane components on the function of AMPs. In this study, we Design of novel antibiotic compounds to overcome the increasing report atomic-level insights into the role of cholesterol on the bacterial resistance towards conventional antibiotics is of consider- function of pardaxin using solid-state NMR experiments on mechan- able current importance [1]. Numerous studies have reported the ically aligned model phospholipid membranes. Cholesterol is abun- isolation, characterization, biological functions, and potential phar- dantly present in mammalian membranes, but not in bacterial maceutical applications of naturally occurring AMPs [2,3]. A variety of membranes, and has been thought to be crucial in reducing the biochemical and biophysical approaches have reported mechanisms toxicity of AMPs. of membrane permeation/disruption, bioavailability, and synergistic Pardaxin is a 33-residue antimicrobial peptide originally isolated activities of AMPs [4–7]. High-resolution cutting-edge techniques from the secretions from the Red Sea Moses sole, Pardachirus have been used to obtain atomic-level insights into the structure, marmoratus. Pardaxin has been shown to exhibit a broad-spectrum dynamics, folding, oligomerization, and membrane orientation of of antimicrobial activities at very low concentrations, to act as a AMPs and their interactions with lipid membrane [8]. These studies channel-forming neurotoxin, and also to cause cell death by necrosis have been utilized in the design of potent peptide antibiotics [9–11]. at slightly higher concentrations [16–21]. Amino acid sequences of In spite of these valuable studies, unfortunately, only a very few pardaxin peptides are given in Fig. 1 along with the 3D NMR structure peptides have been found to be suitable for pharmaceutical appli- of the peptide. An early NMR study on pardaxin in a TFE:water cations [5,12–15]. One of the major difficulties in designing potent solution reported a helix–hinge–helix structure similar to that of AMPs is determining the role of membrane components that play melittin [22]. But a recent NMR study reported the more biologically important roles in the selectivity of AMPs. Therefore, there is a relevant structure of the peptide in a membrane environment, where the structure of the N-terminal domain significantly differs from that obtained from TFE:water structure (Fig. 1) [23]. Recent NMR studies have also reported the membrane orientation, role of membrane ⁎ Corresponding author. Tel.: +1 734 647 6572. composition, and potential mechanisms of the membrane permeation E-mail address: [email protected] (A. Ramamoorthy). of pardaxin [23,24]. The antimicrobial activities of peptide fragments 1 Present address: Department of Fine Chemistry, Seoul National University of corresponding to 1–11, 1–18, 1–22, 10–33, and 12–33 residues of Technology, Seoul, Korea. 2 Present Address: Polymer lab, Central Leather Research Institute, Adayaru, Chennai pardaxin and several mutants have also been reported. The amidated 600 020, Tamil Nadu, India. forms of the 22-residues C-terminal fragment and the 11 amino acid

2. Materials and methods

2.1. Materials

Methanol and chloroform were purchased from Aldrich Chemical Inc. (Milwaukee, WI). Phospholipids, cholesterol, and naphthalene were purchased from Avanti Polar Lipids (Alabaster, AL), Sigma (St. Louis, MO), and Fisher Scientific (Pittsburg, PA), respectively. All these chemicals were used without further purification. All peptides were synthesized and purified as explained in our earlier publication [24]. Mechanically aligned bilayer samples were prepared using the naphthalene procedure as explained in our previous publications [29].

2.2. Solid-state NMR

All of the solid-state NMR experiments were performed on a Chemagnetics/Varian Infinity 400 MHz spectrometer operating at resonance frequencies of 400.138, 161.979, and 40.55 MHz for 1H, 31P, and 15N nuclei, respectively using home-built flat-coil probes. 31P chemical shift spectra obtained using a spin-echo sequence (90°–τ– 180°–τ-acquisition; τ=70 μs) with a 90° pulse length of 3.5 μs under 40 kHz proton decoupling were used to confirm the alignment of each mechanically aligned bilayer samples after an equilibration for about 30 min at a chosen temperature before recording the spectrum. All reported solid-state NMR experiments were performed with the bilayer normal of the sample oriented parallel to the external magnetic field. After optimization of the experimental conditions for sensitivity, resolution, and sample stability using a series of 1D NMR experiments as explained elsewhere [28], 2D BB-PISEMA experiments were performed as explained in our previous publication [26]. A ramp cross-polarization sequence with a 1H π/2 pulse length of 3 μs, 50 kHz cross-polarization power, a 1H decoupling of 75 kHz during acquisition, and a 2 s recycle delay were used in 1D experiments. An RF field strength of 62.5 kHz was used for the Lee–Goldburg off-resonance pulses during the t1 period of BB-PISEMA. 32 t1 increments each with 3600 scans were recorded for 2D BB-PISEMA spectra. All experimental data were processed using the Spinsight (Chemagnetics/Varian) software on a Sun SPARC workstation.

3. Results

A previous solid-state NMR study reported a transmembrane orientation for the C-terminal helix of pardaxin in DMPC and a membrane surface orientation in POPC bilayers [24]. Since we are Fig. 1. Pardaxin—a cationic antimicrobial peptide originally isolated from the Red Sea Moses sole, Pardachirus marmoratus. (A) Amino acid sequences of pardaxin peptides. interested in probing the role of cholesterol on the dynamics of the Pa4 was used in this study. (B) NMR structure of pardaxin determined from DPC peptide and its implications for the barrel-stave mechanism of micelles [23]. (C) A helical wheel representation of the C-terminal amphipathic domain membrane disruption by pardaxin, we chose to use DMPC and of pardaxin. DMPG lipids for this study in which the peptide is in a transmembrane orientation. Two types of model membrane compositions were used in this study: 3:1 DMPC:DMPG bilayers and DMPC bilayers containing N-terminal domain exhibit antimicrobial activity only against Gram- 15 mol% cholesterol to understand the role of cholesterol present in positive bacteria 17], whereas the fragment 1–18 has been shown to mammalian membranes. 2D BB-PISEMA spectra of these samples at be active against Escherichia coli but not against Gram-positive various temperatures are given in Fig. 2. A single peak observed in the bacteria [25]. These differences in the activities of the derivatives of 15N chemical shift region of 175 to 183 ppm and a 15N–1H dipolar pardaxin are not well understood. coupling frequency of 8–9.3 kHz confirms the transmembrane In this study, we show that the presence of cholesterol reduces the orientation of the C-terminal helix of pardaxin under this condition. dynamics of pardaxin but does not influence the membrane 2D experiments were performed to measure the influence of orientation of pardaxin. Influences of temperature and cholesterol temperature in the presence and absence of cholesterol. A decrease on the backbone dynamics of pardaxin are investigated using a in both the 15N chemical shift and 15N–1H dipolar coupling combination of 2D broadband-PISEMA (polarization inversion spin frequencies were observed with increasing temperature in the exchange at the magic angle) experiments and mechanically aligned absence of cholesterol (Fig. 3A). On the other hand, no significant phospholipids bilayers to accurately measure 15N chemical shifts and changes were observed in the presence of cholesterol (Fig. 3B). A 1H–15N dipolar couplings [26–28]. Implications of solid-state NMR model explaining the influence of cholesterol on lipid bilayers and on results on the mechanisms of membrane disruption and selectivity by pardaxin's backbone dynamics is depicted in Fig. 4 and discussed pardaxin are discussed. below. A. Ramamoorthy et al. / Biochimica et Biophysica Acta 1798 (2010) 223–227 225

Fig. 2. The C-terminal helical segment of pardaxin has a transmembrane orientation. 2D BB-PISEMA spectra of mechanically aligned lipid bilayers containing 2 mol% 15N-Ala21- pardaxin: (top) 3:1 DMPC:DMPG bilayers and (bottom) DMPC bilayers with 15 mol% cholesterol at 30, 37 and 43 °C. A single peak observed in each spectrum, even in the absence of cholesterol, suggests an average of dynamically disordered tilts of the C-terminal helical domain of pardaxin.

4. Discussion the geometry of toroidal pores induced by antimicrobial peptides was also determined at high resolution [42,43]. Previous studies have Previous solid-state NMR studies have shown that pardaxin has a shown that the absence of the overall motion is an advantage in the transmembrane orientation in DMPC bilayers while it is oriented close structural studies of transmembrane proteins [30]. Lack of bulk water to the bilayer surface of POPC bilayers due to the hydrophobic in mechanically aligned bilayers enabled the measurement of mismatch between its hydrophobic length and the hydrophobic backbone dynamics only from membrane-bound peptide populations. thickness of the lipid bilayer [23,24]. In this study we measured the Therefore, in this study, we used this approach to determine the backbone dynamics of the C-terminal amphipathic helix of pardaxin influence of the backbone dynamics on the function of pardaxin. using solid-state NMR experiments on mechanically aligned lipid Pardaxin was labeled with 15N at the amide site of Ala21 residue, bilayers at various temperatures. Dipolar couplings between 15N and which is located in the helical region of the peptide (Fig. 1B) [23].A 1H nuclei at the amide site of pardaxin selectively labeled with 15N single peak observed near the parallel edge of 15N chemical shift isotope were accurately measured using 2D broadband-PISEMA anisotropy with an N-H dipolar coupling ∼8 kHz in 2D PISEMA (Fig. 2) experiments. The influence of cholesterol embedded in lipid bilayers spectra suggest that the C-terminal helix has a transmembrane on the dynamics of pardaxin was also investigated. orientation confirming previously reported channel-forming mechanism of Pardaxin [23,24]. The variation of NMR parameters with 4.1. PISEMA experiments on mechanically aligned lipid bilayers provide temperature is given in Fig. 3 and discussed below. piercing insights into pardaxin's function 4.2. Cholesterol reduces pardaxin's dynamics and disorder in its A number of studies have well utilized a combination of PISEMA transmembrane orientation and mechanically aligned lipid bilayers to investigate folding and topology of membrane-associated peptides and proteins [30–36]. This Our previous solid-state NMR study could not differentiate the approach has also revealed mechanisms of membrane disruption by effects of the tilt of the C-terminal amphipathic helix from the bilayer antimicrobial peptides [37–40] and amyloid peptides [41]; and in fact normal and the peptide's dynamics on the observed 15N chemical shift frequency from mechanically aligned bilayers [24]. In this study, we measured 15N chemical shift and 1H–15N dipolar coupling values by varying the temperature. As seen from 2D PISEMA data in Fig. 2, both chemical shift and dipolar coupling values decrease as the temperature of the sample is increased. These results suggest that the transmembrane orientation of the peptide is disordered due to dynamics. Increasing temperature increases the mobility of the transmembrane C-terminal helix rendering the peptide to orient heterogeneously relative to the bilayer normal. On the other hand, a decreasing temperature reduces the mobility of the helix and also the disorder in the tilt of the helix. Our results in Figs. 2 and 3 indicate that the presence of cholesterol decreases the disorder in the tilt of the C- terminal helix and also its mobility even at 43 °C. This is mainly Fig. 3. Cholesterol reduces pardaxin's dynamics in lipid bilayers. Changes in 15N because cholesterol increases the order of acyl chains of the lipid chemical shift (A) and 1H–15N dipolar coupling (B) frequencies measured from 2D BB-PISEMA spectra of 3:1 DMPC:DMPG bilayers (open circles) and DMPC bilayers with bilayer, which restricts the mobility of the C-terminal helix of the 15 mol% cholesterol (filled triangles). peptide (Fig. 4). These observed changes in the mobility of the 226 A. Ramamoorthy et al. / Biochimica et Biophysica Acta 1798 (2010) 223–227

Fig. 4. A model depicting the dynamics-driven membrane orientation of pardaxin. (A) Disordered lipid acyl chains thin the bilayer and therefore disordered tilted orientations are expected for the C-terminal helix of pardaxin. (B) The presence of cholesterol thickens the lipid bilayer, reduces the dynamical disorder in the transmembrane region, and could reduce the channel activity and hemolytic activity of pardaxin.

channel-forming helix and also in its transmembrane orientation suppress the channel-forming activity of pardaxin and therefore due to the presence of cholesterol or because of a change in the would play a role in the selectivity of this antimicrobial peptide. This temperature suggest that peptide-peptide interactions are not prediction from our studies enables us to hypothesize that any sufficiently strong to retain the same tilt angle of the helix. This is in mutation of pardaxin that is accompanied by a reduction in the excellent agreement with the previously reported difference in the dynamical disorder of the C-terminal helix would reduce the membrane orientation of the C-terminal helix in DMPC vs. POPC hemolytic activity of the mutant peptide. For example, a substitution bilayers due to hydrophobic mismatch [23,24]. These results therefore of Ala for Pro-13 should extend the helicity towards the N-terminal imply that cholesterol significantly influences the barrel-stave domain by removing the hinge and therefore should significantly mechanism of pardaxin; similar effects have been reported for other suppress the dynamical disorder in the C-terminal transmembrane channel-forming peptides like melittin [24,44–50]. Since cholesterol helical region of the mutant. Such Pro to Ala mutation has indeed been is abundantly present in mammalian cell membranes, it would be reported to abolish the toxicity of the peptide [7]. It is also interesting interesting to investigate the effect of higher concentration of to note that similar behavior has been reported for a derivative of cholesterol on the barrel-stave activities of pardaxin. A higher pardaxin that lacks the first 9 N-terminal residues [7]. Since this concentration of cholesterol could prevent the membrane insertion derivative lacks the N-terminal domain and the disorder in its of pardaxin [16]. It is also be possible that the direct interaction membrane orientation could be smaller, its hemolytic as well as between cholesterol and pardaxin could play some role in the antimicrobial activities are considerably reduced. Therefore, there is a observed dynamical changes or tilt of the helix, particularly at a clear correlation between the dynamics-induced heterogeneity in the concentration of cholesterol higher than used in this study. More tilt of the C-terminal helix and the barrel-stave mechanism of solid-state NMR experimental measurements are needed to confirm pardaxin. Therefore, we believe these finding will be useful in the this effect. 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